TRPV1 ion channels are multimodal receptors that can be activated by heat, high [H+]o, voltage, arachidonic acid metabolites, capsaicin (the pungent extract of hot chili peppers), and the signaling lipid PI(4,5)P2 (PIP2). Ca2+/Calmodulin (Ca2+/CaM) and ATP may modulate its activity as well. Our long-term goal is to understand the molecular mechanism by which TRPV1 integrates these multiple physiological stimuli. We and others have previously established that PIP2 directly activates TRPV1. Our recent work indicates that the proximal part of the intracellular C-terminal domain comprises at least part of the PIP2 binding site. However, the inability to control the lipid composition of native membranes, the presence of myriad enzymes and other proteins in cells and excised patches, and the difficulty of specifically labeling intracellular domains of channels within cells have proven serious experimental barriers to understanding regulation of TRPV1 by PIP2 and other activation modalities. We have developed a novel approach to reconstitute purified TRPV1 channels at high density in synthetic Giant Unilamellar Vesicles (GUVs). In this proposal we will apply standard patch-clamp methods, Patch-Clamp Fluorometry (PCF), and Transition Metal Ion FRET (tmFRET) to study purified TRPV1 channels in GUVs of defined lipid composition. Single cysteines engineered into our cysteineless TRPV1 background will be used to site-specifically label channels in the GUVs with fluorophore, completely eliminating the background fluorescence problem. The GUVs used for reconstitution will include synthetic lipids that bind transition metals which act as short- distance FRET quenchers in the novel short-range tmFRET approach we have developed. PCF allows us to simultaneously record the function of the channel with electrophysiology and the rearrangement of the channel with fluorescence. These new tools will allow us to measure dynamics of the intracellular N- and C-terminal domains associated with PIP2 activation as well as with activation by heat, Ca2+/CaM, and ATP.